Calorimetry in Biotechnology

### **Chapter 7** Calorimetry to Quantify Protein-Ligand Binding

*Salerwe Mosebi*

#### **Abstract**

Isothermal titration calorimetry (ITC) is the preferred method used to study biochemical reactions like protein-ligand binding due to its sensitivity, accuracy, and precision. ITC measures directly the heat absorbed or released (∆*H*) associated with a given binding process. A typical ITC experiment allows the dissection of the binding energy of a reaction into ligand-enzyme association constant (*Ka*), change in enthalpy (∆*H*), change in entropy (∆*S*), change in Gibbs-free energy (∆*G*), and the stoichiometry of association (N). The change in heat capacity (∆*Cp*) is obtained from the measurements of binding enthalpy over a range of temperatures. The magnitude and signs of the thermodynamic parameters that were obtained provide insight into the nature of interactions involved in the binding process. The strength of interaction is thermodynamically favorable is determined by the Gibbs free energy. ∆*G* is an important thermodynamic descriptor of a binding reaction since it dictates the binding affinity and is in turn defined by the enthalpy and entropy changes expressed in the following equation: ∆*G* = ∆*H*–*T*∆*S*. Up-close, this reflects the contradistinctions of two thermodynamic effects at a molecular level—the propensity to drop to lower energy (bond formation, negative ∆*H*), counterbalanced by the innate thermal Brownian motion's destructive characteristic (bond breakage, positive ∆*S*).

**Keywords:** isothermal titration calorimetry, binding energy, association constant, entropy change, enthalpy change, heat capacity

#### **1. Introduction**

The completion of the human genome project over 18 years ago has catapulted the number of novel targets for drug development to great heights. Many of these targets belong to protein families with homologous structures and similar binding pockets, which are crucial in regulating pathways and interaction networks describing cell function and inter-relation. It is also apparent that the basis of molecular recognition in drug discovery, signal-transduction, and protein-ligand complexes requires complete structural and thermodynamic dissection of macromolecular interactions involved. Several techniques (fluorescence, absorbance, nuclear magnetic resonance, surface plasmon resonance, biolayer interferometry, and ultracentrifugation) have been used as premier tools for characterizing interactions of biomolecules. These techniques can only determine the binding affinity constant (*K*a) and indirectly derive other thermodynamic parameters. However, due to its sensitivity, accuracy,

and precision, isothermal titration calorimetry (ITC) is the most rigorous and preferred method applied in a wide range of chemical and biochemical reactions. ITC has the advantage of directly quantifying the binding energetics of biological processes that include but not limited to protein–protein binding, protein-ligand binding, protein-DNA binding, protein-carbohydrate binding, protein-lipid binding, and antigen–antibody binding. ITC does this by measuring, directly and in realtime, the heat absorbed or released (∆*H*) associated with a given binding process. A typical ITC experiment allows the dissection of binding energy of a reaction into ligand-enzyme association constant or binding affinity (*K*a), change in enthalpy (∆*H*), change in entropy (∆*S*), change in Gibbs free energy (∆*G*), the stoichiometry of association or number of binding sites (N), and the change in heat capacity (∆*C*p) obtained from measurements of binding enthalpy over a range of temperature. More importantly, ITC can be used to determine very low (103 M−1) to very high association constants (1012 M−1) without the need to use labels or immobilization of the binding components.

### **2. Fundamental principles of the ITC technique**

A detailed description of the instrument and technique can be found elsewhere in the literature [1–5]. Briefly, the titration calorimeter consists of the injector system, adiabatic shield, and matched reference and sample cells (see **Figure 1a)**. There is a self-stirring padded injection syringe and the thermostatic and feedback power systems that are computer-controlled. This instrument measures in real-time the thermal power that occurs when a solution in a syringe is titrated into a sample cell. In a typical ITC instrument, a pair of cylindrical cells (referred to as sample cell and reference cell) with volumes ranging from 200 to 1400 μl are present and contain analyte solution and reference buffer or water, respectively [6, 7]. The

#### **Figure 1.**

*(***A***) A schematic representation of the ITC instrument setup, showing the sample and reference cells enclosed by a thermostated jacket. (***B***) an example of an ITC assay.*

#### *Calorimetry to Quantify Protein-Ligand Binding DOI: http://dx.doi.org/10.5772/intechopen.102959*

thermostated adiabatic shield ensures that no heat exchange occurs between the cells and the surroundings [2]. The two cells are maintained at a constant and identical temperature by a feedback system that supplies thermal power continuously. In the event of a reaction in the sample cell usually accompanied by heat (exothermic reaction), the system ensures that the feedback power is withdrawn in order to retain thermal equilibrium between the cells. The feedback power supplied or withdrawn by electric resistive heaters located on the outer surfaces of the sample and reference cells to minimize temperature imbalances upon ligand injection is measured and converted into the heat of interaction. A sequence of injections is programmed and the ligand solution is injected at regular intervals into the sample cell through an automated injection syringe, which is stirred by rotation of the paddle-shaped syringe. After each injection (typically between 1 and 20 μl), the composition inside the sample cell changes causing the rearrangement of populations and complex formation [5]. Accordingly, as the series of injections continues, the system will experience various states of equilibrium each differing in composition. The heat released or absorbed with each injection corresponds to the increase in interacting species' concentration (as the reaction advances), and it is determined by the integration of the region under the deflection signal measured (amount of heat per unit of time provided to maintain thermal equilibrium in the sample and reference cells) [5]. If the binding between the injectant and the analyte is exothermic, this will result in the reduction in the power supplied by the feedback heater to maintain a constant temperature. On the other hand, if the binding is endothermic, there will be an increase in feedback power. At the end of the experiment, when no further heat is released or absorbed in the sample cell and saturation of the macromolecule is reached and it is possible to estimate *K*a, ∆*H*, and N (independent variables). A typical result output of an ITC instrument is the feedback power measured as a function of time as shown in **Figure 1b**. The top panel represents the sequence of peaks as the solution in the syringe is injected into the analyte in the sample cell. The observed signal is the additional power that needs to be supplied or removed during the course of the experiment to keep a constant temperature in the sample cell and equal to the reference cell temperature. The reaction shown is that of an endothermic reaction, with an integrated heat plot in the bottom panel. Consequently, the areas under each peak, derived from per mole of ligand injected in each injection, are then plotted against the molar ratio of the total concentration of ligand to protein molecule concentration in the sample cell to obtain the following independent thermodynamic parameters: binding affinity, binding enthalpy, and the stoichiometry of binding. Notably, if two binding processes are characterized by different enthalpic and entropic terms and have the same Gibbs free energy of binding, they correspond to different binding modes, and therefore, the main underlying intermolecular interactions are different.

#### **3. Protein-ligand binding energetics**

As mentioned before, a typical ITC experiment allows the thermodynamic dissection of binding energy of a reaction into (*K*a), (∆*H*), (∆*S*), (∆*G*), (*N*), and (∆*C*p). Importantly, the magnitude and signs of the thermodynamic parameters obtained give us clues into the nature of interactions involved in the binding process, for example, the strength of the interaction and whether or not it is thermodynamically favorable is determined by the Gibbs free energy. If one ponders on the binding

reaction under equilibrium conditions, where a macromolecule (P, protein) binds another molecule (L, ligand):

$$\text{P} + \text{L} \leftrightarrow \text{PL} \tag{1}$$

And if you assume that only one binding site is available, the association constant, *K*a, which is inversely proportional to the dissociation constant, *K*d, one is then able to determine the partition of the reactant molecules into free and bound species according to Eq. (2) below:

$$K\_x = \lceil PL \rceil / \lceil \mathbf{P} \rceil \lceil \mathbf{L} \rceil \tag{2}$$

The Gibbs free energy change of binding is an important thermodynamic descriptor of a binding reaction since it dictates the binding affinity or association constant:

$$
\Delta G = -\text{RTl} \ln K\_r \tag{3}
$$

where R is the universal gas constant (8.314 J/mol/K), T is the temperature in kelvin, and *K*a is the equilibrium binding constant. ∆*G* is in turn defined by the enthalpy and entropy changes and is expressed in the following equation:

$$
\Delta G = \Delta H - \text{T}\Delta S \tag{4}
$$

At the molecular level, this reflects the contradistinctions of two thermodynamic effects at a molecular level—the propensity to drop to lower energy (bond formation, negative ∆*H*), counterbalanced by the innate thermal (Brownian) motion's destructive characteristic (bond breakage, positive ∆S) [8].

Since the native state of a protein exists as an ensemble of conformational states, the energy of stabilization of protein structure will not be evenly distributed throughout its three-dimensional structure [9]. There are regions of the protein with high stability constants (e.g., the hydrophobic core) and regions with low stability constants (e.g., loops and turns) with the majority of proteins exhibiting a dual character as originally observed for the HIV-1 protease [10, 11]. Since ligands with low molecular weight are in general not found attached to the exterior of proteins but are engulfed in crevices or binding pockets created by loops or other proteins' structural elements, the number of interactions between ligand and protein is increased and concomitantly enshrouds a substantial surface area from the solvent [9]. This conformational rearrangement often permits the entry of the ligand into the binding site and its subsequent shielding from the solvent; hence, makes favorable contributions to the Gibbs free energy of binding. If the rearrangements are only transient and the free and the bound states of the protein are similar, only binding kinetics are affected. If, however, the free and bound conformations of the protein are different, the binding affinity will be affected [9]. The Gibbs free energy associated with the change in protein conformation from its free to its bound state is included in the computation of the effective Gibbs energy of binding and corresponding binding affinity:

*Calorimetry to Quantify Protein-Ligand Binding DOI: http://dx.doi.org/10.5772/intechopen.102959*

$$
\Delta G\_{\text{bind}} = \Delta G^{\circ}\_{\text{bind}} + \Delta G\_{\text{conf}} \tag{5}
$$

where ∆*G°*bind is the Gibbs energy of binding obtained under the assumption that the free and bound states of the protein are the same, and ∆*G*conf is the Gibbs energy associated with the change in protein conformation from its free to its bound form. In general, the Gibbs energy associated with a change from a less stable region to the bound conformation will be smaller than that associated with a change from a stable conformation to the bound conformation [9]. The presence of flexible regions in the protein molecule appears to facilitate the ligand-induced conformational changes if the putative binding site is not binding-competent in the ligand-free protein. The presence of regions with low stability also appears to provide a mechanism for achieving high binding affinity for low molecular weight ligands and serves as a starting point for the propagation of binding signals to distal sites [9].

Enthalpic and entropic contributions of the Gibbs energy originate from different types of interactions in the binding process. The binding enthalpy primarily reflects the energetic contribution of many individual interactions (hydrogen bonds, van der Waals interactions, polar, and dipolar interactions) between the ligand and the protein during the binding process, the conformational changes associated with binding, including interactions associated with the solvent. A negative (favorable) ∆*H* occurs when the interactions between the interacting molecules (e.g., hydrogen bond formation and van der Waals interactions) over-compensate the interactions of the individual molecules with the bulk solvent; otherwise, it will be positive (unfavorable, as for nonspecific hydrophobic interactions) [12]. The observed binding ∆*H* measured from a single ITC experiment often includes contributions not only from the actual binding event but also from the heat that is due to buffer ionization [13–15]. This is particularly true when the primary binding event is accompanied by the transfer of protons between the solvent and the protein-ligand complex. Thus, the determination of the intrinsic energetics of ligand binding requires experiments or measurements to be performed separately as a function of pH in buffers with different ionization enthalpies [13, 14, 16]. From this, p*K*a values of ionizable groups responsible for proton linkage in the free and bound states and the number of protons that are coupled to the binding reaction can be easily calculated [14].

The binding entropy refers to the degree of disorder accompanying complex formation. Two major terms that contribute to the change in entropy are the solvation and conformational entropies. Solvation entropy arises from the gain in degrees of freedom of water molecules that, prior to the binding, are localized on the surface of the binding molecules and are released to the bulk solvent upon binding due to partial or complete desolvation of the two binding molecules. The change in solvation entropy is favorable (positive) if the surfaces that are buried upon binding are predominantly hydrophobic. It, therefore, originates from the burial of hydrophobic surfaces upon binding. Entropically driven ligand binding reactions are characterized by a large positive entropic contribution driven by the tendency of the molecule to escape water rather than by favorable interactions with the target molecule. In addition, the burial of solvent-exposed molecular surface area upon binding also contributes substantially to the heat capacity change upon complex formation due to the release of electro-restricted water or "hydrophobic water" from the binding site [17]. The conformational entropy, on the other hand, arises from changes in conformational degrees of freedom experienced by both the protein and the ligand upon binding. It is usually negative (unfavorable) due to the loss of degrees of freedom

resulting from the reduction in the number of accessible conformations and configurations of both molecules (protein and ligand) upon binding.

#### **4. Protein-ligand quantification and lead drug design**

Currently, the development of lead compounds or drug design is centered on the optimization of their binding affinity toward the intended target. The binding affinity of a compound can be improved by generating a favorable binding enthalpy, favorable solvation entropy, and by minimizing the unfavorable conformational entropy. It is evident that simultaneous optimization of the three factors can achieve extremely high affinity. However, it is entirely feasible to design lead compounds that bind to the intended target with similar affinity but with different binding mechanisms, i.e., entropically or enthalpically driven ligands [18]. Entropically driven ligand derives most of its binding energy from a nonspecific hydrophobic effect, i.e., by making interactions of the drug with the solvent unfavorable, whilst enthalpically driven ligand derives its binding energy by establishing strong and specific hydrogen bonds with the target. Drug designers have long aimed at developing conformationally constrained ligands preshaped to the geometry of the selected binding site, which completes entropy optimization. Accordingly, a conformationally constrained molecule that is preshaped to the target achieves affinity, specificity, and selectivity through hydrophobicity and shape complementarity [19]. Perhaps, the most significant example is given by the development of the first-generation HIV-1 protease (HIV-1 PR) inhibitors (saquinavir, ritonavir, indinavir, and nelfinavir). The binding of these HIV-1 protease inhibitors is entropically driven and their binding enthalpy is either unfavorable (saquinavir, indinavir, and nelfinavir) or only slightly favorable (ritonavir) [20, 21]. In all cases, the dominant force for binding is a large positive entropy change that originates primarily from the burial of a large hydrophobic surface upon binding [20]. Moreover, since shape and hydrophobicity are nonspecific interactions, a change in the target binding site would lead to a reduction in the binding affinity. A low binding affinity reflects the inability of these conformationally rigid ligands to adapt to changes in the target binding pocket due to mutations or naturally occurring polymorphisms arising from genetic diversity. Hydrophobicity has historically been the preferred variable in the pharmaceutical industry due to its ease of implementation [22].

An enthalpically driven binding indicates specific interactions between two binding partners and corresponds well with ligand specificity, selectivity, and adaptability. Alternatively, an unfavorable enthalpic binding energy is indicative of nonspecific interactions between the binding partners, which in turn affects the ligand's specificity, selectivity, and adaptability. Despite apparent advantages of enthalpic interactions in achieving high affinity and improved selectivity, the optimization of the binding enthalpy has been more cumbersome to implement due to a large and unfavorable desolvation enthalpy of polar groups [23]. Generally, a polar group needs to make a strong interaction with the target in order to compensate for the desolvation enthalpy. Energetic contributions to binding affinity are not simply localized to the direct interactions between the molecules but contain interactions from structural and dynamic changes propagated throughout the protein, and from counter ions and hydrating water molecules located at the binding site. To be effective, an inhibitor needs to exhibit an extremely high affinity for the intended target and be mildly affected by

#### *Calorimetry to Quantify Protein-Ligand Binding DOI: http://dx.doi.org/10.5772/intechopen.102959*

mutations. Ideally, an inhibitor should have a binding affinity in the 1–50 pM range against the wild-type and be affected by mutations by a factor of 100 or less [24–26]. Compounds that achieve high binding affinity or that maximize binding affinity have been shown to combine or balance the favorable entropic and enthalpic contributions to the overall Gibbs energy of binding [27–30].

Notably, drug design paradigms have, to a large extent, illustrated how the enthalpic or entropic character of inhibitors is not dependent on the intended target, and that it is possible to develop entropically as well as enthalpically optimized inhibitors against the same binding site (e.g., HIV-1 protease). It has, for example, taken over 10 years to optimize HIV-1 protease inhibitors from the entropically driven inhibitors to the new and more potent enthalpically driven inhibitors [21, 24, 31]. The second-generation HIV-1 protease inhibitor, KNI-764 (AG-1776) for example, achieves the highest affinity (*K*d = 32 pM) to the HIV-1 protease with a binding enthalpy (∆*H*) of—7.6 kcal/mol and an entropic contribution (−T∆*S*) of −6.7 kcal/mol and can still afford the presence of certain flexible elements [21, 32]. The introduction of flexible asymmetrical functional groups in regions facing or in close proximity to mutation-prone areas of the protein provides adaptability to the inhibitor and low susceptibility to mutations [25, 26]. The increased conformational flexibility found in the second-generation HIV-1 protease inhibitors can also allow the inhibitor to compensate for the loss of interactions as a result of mutations in the target by burying a comparable or even larger surface area from the solvent [25].

New drug design strategies by calorimetric characterization have permitted the designers to recognize the nature of forces by which the HIV-1 proteins inhibitors bind the target primarily because these forces originate from different interactions. ITC was particularly crucial at the later stages since it gave a detailed description of the thermodynamic factors governing protein-inhibitor interactions essential for molecular recognition in HIV-1 protease binding and led to improvement in drug design. This task was also facilitated by structure-based algorithms able to predict the enthalpic and entropic consequences of introducing different functional groups in the lead compounds under investigation [9, 32]. Extensive studies using numerous techniques of molecular biology and the deepened understanding of drug-target at the molecular level have helped greatly in achieving rapid success in the area of drug development, especially in the treatment of AIDS [33–42].

#### **5. Experimental approaches to determining the protein-ligand binding energetics using ITC**

ITC experiments can be performed to determine the binding affinity, binding enthalpy, Gibbs free energy of binding, and stoichiometry of different inhibitors to the wild-type HIV-1 (South African subtype C (C-SA) protease. Indinavir, used in this study as an example, is an inhibitor that binds the wild-type HIV-1 protease with high affinity (with *K*i ≤ 1 nM). Therefore, the typical titration experiments are not able to accurately determine the association constant, even though the enthalpic contributions can be measured with high precision. A solution to this challenge is to perform calorimetric displacement titrations that will allow for the calculation of the binding affinity and enthalpy, as reported previously [25, 26, 43]. This technique allows complete determination of binding thermodynamics of high-affinity ligands (*K*a ≥ 109 M−1) that are beyond the range of determination by direct titration. In calorimetric displacement

titrations, the high-affinity inhibitor is titrated into a protease sample prebound to a weaker binding inhibitor (acetyl-pepstatin), a well-characterized inhibitor of lower binding affinity and unfavorable binding enthalpy [11]. The selection of a weak binding inhibitor with a binding isotherm of opposite sign (positive ∆*H*) produces a larger signal during the displacement reaction due to the displacement of the weaker binding inhibitor by an inhibitor yielding an exothermic isotherm (negative ∆*H*). As depicted in **Figure 2**, in the presence of the weak binding inhibitor, the apparent binding constant for the inhibitor which binds tightly, *K*app, falls within the range required for ITC determination. *K*app is given by Eq. (6) below:

$$Kapp = K \mathbf{a} \;/\left(1 + K\mathbf{B}\lceil\mathbf{B}\rceil\right) \tag{6}$$

where B is the concentration of the weaker binding inhibitor. In addition, *K*app can be lowered to the desired level by increasing the concentration of the weak

#### **Figure 2.**

*Overview of a displacement titration assay for HIV-1 protease. The binding affinity of ritonavir, Ka, is beyond the limit of direct calorimetric determination. The displacement titration experiment is performed in the presence of the weak binding inhibitor acetyl-pepstatin (KB = 2.0 × 106 M−1).*

#### *Calorimetry to Quantify Protein-Ligand Binding DOI: http://dx.doi.org/10.5772/intechopen.102959*

inhibitor. In addition, the binding isotherm, in this case, has sufficient curvature to allow for the calorimetric measurement of the apparent binding affinity of the stronger binding ligand [43]. Two calorimetric titrations need to be performed to work out the binding competition equations and calculate the association constant and enthalpic contributions of the tight-binding inhibitor: (1) titration of the weak binding inhibitor into the protease and (2) titration of the inhibitor of interest into the protease-(weak binding inhibitor) complex. The competition experiments were also performed at pH 5.0 using an acetate buffer with negligible binding enthalpy to minimize any proton coupling effect on the observed binding enthalpy [25].

**Figure 3** shows typical displacement titrations for active site inhibitors of the wild-type C-SA HIV-1 protease in the presence of acetyl-pepstatin, pH 5.0. Each peak in the top panel represents the displacement of a weaker binding inhibitor (acetyl-pepstatin) from the active site of the protein by the tight-binding inhibitor (e.g., indinavir, with high binding affinity). As the titration progresses, the area under each peak becomes smaller due to increased occupancy of the available binding sites on the enzyme by the inhibitor of interest. The bottom panel in the figure shows the calorimetric binding isotherm obtained by plotting integrated heats obtained after each injection as a function of inhibitor concentration of interest per protein dimer. **Figure 3a** shows the integrated heats for the above peaks plotted against the molar ratio of acetyl-pepstatin to HIV-1 protease molecule. The solid line through the data represents the best fit using a one-site binding model. For the wild-type HIV-1 C-SA protease, the experimental data fit best to a

#### **Figure 3.**

*(A) A representative calorimetric profile of the titration of the wild-type HIV-1 C-SA protease with acetylpepstatin. Titrations of acetyl-pepstatin (300 μM) into protease solution (20 μM). (B) ITC displacement calorimetric titration of indinavir (250 μM) into a solution of the wild-type HIV-1 C-SA protease (20 μM) prebound to acetyl-pepstatin (200 μM).*

single-site displacement binding model; i.e., with the stoichiometry of 1:1 as shown in **Figure 3b**. The binding isotherms are monophasic with a sigmoidal fit to the data representing the decrease in available binding sites on the protein as the reaction progresses to completion. Used as a reference here, the clinical inhibitor, indinavir, binds to the wild-type C-SA HIV-1 protease with high binding affinity, *K*a, of 0.2 × 10<sup>9</sup> M−1 in a process strongly favored by entropic contributions, contributing about 12 kcal/mol to the overall Gibbs energy of binding at 25°C. At 25°C, the stoichiometry of binding for indinavir is 1.0 interpreted as one molecule of the inhibitor bound per dimer of HIV-1 protease and is consistent with crystallographic data [44–48]. Accordingly, the binding of indinavir is favored by entropic contributions of −15.0 kcal/mol, whereas its binding to the wild-type HIV-1 C-SA protease is characterized by a positive (unfavorable) enthalpy change of 2.70 kcal/mol. This is in agreement with the thermodynamic data obtained previously, which showed entropically controlled binding affinities and unfavorable or slightly favorable binding enthalpies [20, 49, 50]. Interestingly, for indinavir and other HIV-1 protease inhibitors like saquinavir, ritonavir, and nelfinavir, entropy (−T∆*S*) contributions as large as −16 kcal/mol, have also been shown by others to be required to compensate for the unfavorable binding enthalpies [20, 49].

#### **6. Determination of the heat capacity change of a binding reaction**

Although with a single ITC experiment, one is able to gain insights regarding the binding constant, binding enthalpy, binding entropy, stoichiometry of the reaction, and the Gibbs free energy of binding, another important parameter—the change in heat capacity (Δ*C*p) upon binding can be obtained by performing the experiment at different temperatures and constant pressure. By applying Eq. (7) below, one can determine its value:

$$
\Delta \mathbf{C}\_{\rm o} = \left( \partial \Delta \mathbf{H} \, / \, \partial \mathbf{T} \right)\_{\rm o} \tag{7}
$$

where *H* is the enthalpic change of binding at different temperatures (T) and Δ*C*p is the change in heat capacity or slope obtained from plotting Δ*H* versus temperature. The heat capacity of a binding reaction is indicative of the burial of polar and nonpolar surfaces upon binding [51–53]. Δ*C*p on an ITC instrument is typically obtained by measuring the enthalpic contributions of binding from 10–35°C at 5°C intervals without changing buffer and pH conditions. Although reports of the binding processes between a protein and a ligand have shown a negative and < 1 kcal/Kmol Δ*C*p, the binding of two macromolecules (e.g., antigen–antibody) can induce higher heat capacity change, which is reflective of the burial of a larger solvent-accessible surface area as a result of the binding [26].

#### **7. Conclusions**

This chapter demonstrated the important role of calorimetry, in particular, isothermal titration calorimetry in dissecting the binding profile of two interacting species (e.g., a macromolecule and a ligand). It has obvious applications in drug development as it can be used for the characterization and optimization of lead compounds due to a wealth of thermodynamic information that is obtained from a

*Calorimetry to Quantify Protein-Ligand Binding DOI: http://dx.doi.org/10.5772/intechopen.102959*

single experiment. Some of the notable successes are in the lead optimization of HIV drugs exemplified by the HIV-1 protease discussed above. To this day, ITC remains a favored technique that can accurately characterize the interaction between the macromolecules and their biologically relevant binding partners. It is also uniquely positioned to assist us in getting a deepened thermodynamic understanding of the important biological processes in living systems like metabolism, active transport, biosensing, regulation, signal transduction, and integration to name a few.

#### **Acknowledgements**

The author would like to acknowledge the University of South Africa and the University of the Witwatersrand for the financial assistance and provision of resources and infrastructure needed to complete this work. The work was also supported by a grant from the National Research Foundation (Grant 121281 to S.M). Lastly, the author would like to thank Prof. Yasien Sayed from the University of the Witwatersrand for the invaluable supervisory role he played on the project.

#### **Conflict of interest**

The author declares no conflict of interest.

#### **Author details**

Salerwe Mosebi University of South Africa, Florida, South Africa

\*Address all correspondence to: mosebs@unisa.ac.za

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 8** Calorimetry in Allergy Diagnostic

*Evgeni Stanev and Maria Dencheva*

#### **Abstract**

Calorimetry is an indisputable diagnostic method. Over the years, there has been an improvement in the equipment and methods for measuring the calor that accompanies various processes. Using a thermal camera, we can measure the surface temperature of the skin at the beginning and the end of each skin allergy test. They are epicutaneous, cutaneous, and percutaneous. In case of a positive reaction, allergic inflammation is observed with the obligatory symptoms, one of which is warming (calor). Measuring and visualizing this warming is essential in the diagnosis of allergic reaction. The methodology of imaging the skin areas and processing the results is the key point in the objectivity of the study. Diagnostic skin allergy tests report mainly immunopathological reactions of the first and fourth types (Coombs and Gel classification). Their course is different and this necessitated the development of various thermovisiographic imaging methods. Through the results of our thermal imaging studies, we derived a scale, that determines the intensity of the allergic reaction, for each of the skin allergy tests. The use of thermovisiography in addition to the standard reporting of allergic skin reactions provides precision and more information about the subtle temperature changes that accompany allergic reactions.

**Keywords:** allergic inflammation, thermocamera, local anesthetics, allergens, haptens, prick test, patch test, intradermal test

#### **1. Introduction**

Diagnosis in allergology is based on anamnestic data, clinical and laboratory tests. Despite the development of *in vitro* tests, skin allergy tests are used as the gold standard in clinical practice. They aim to provoke a local allergic reaction in the study area. Allergic inflammation is characterized by the same five signs of inflammation—tumor, rubor, calor, dolor, and functiolesa. In standard clinical practice, the strength of the reaction is determined by the size of the erythema (rubor) and the size of the papule (tumor). With the help of modern thermal imaging cameras, it is possible to take into account another parameter of the inflammatory reaction—calor. The warming covers the area of skin that has been in contact with the allergen or the hapten.

Skin allergy tests are easy to perform, inexpensive, quick to read, and ideal for diagnosis even in patients with limited mobility, but standard reporting carries with it a degree of subjectivity.

Methodology and implementation of the test:

The test is usually performed on the volar side of the forearm skin according to the skin prick test—European standards. The skin is cleansed with an alcohol swab. The

places of application of the allergen—most often inhaled household, pollen allergens, insect and food allergens, some drugs, local and general anesthetics, iodine contrast agents, and vaccines—are marked with a skin marker. Against each marking is placed one drop of the substance. With a separate plastic lancet, the skin is pricked. Positive and negative controls are obligatory in this test. Conventional reading is after 20 minutes [1].

Method of performing an intradermal test:

In the intradermal test, a certain amount of the allergen is injected into the deeper skin layers. The reading is also 20 minutes [1].

The results of controlled skin sensitization in Prick and the intradermal test are reported by measuring in millimeters the diameters of the papule and erythema that form.

Methods of epicutaneous testing:

Testing with hypoallergenic patches, which are most often placed on the back. This is a test method for proving contact allergy/contact dermatitis, metal allergy, drug allergy, dental materials. The set of allergens for epicutaneous testing is applied separately in special chambers on the test patches, then glued to the back for a period of 48 hours.

It is reported to be a cell-mediated mechanism (Coombs and Gell type 4) of reaction on the 3rd and sometimes on the 7th day [2, 3].

The clinician needs to have accumulated practical knowledge and experience to be able to correctly report skin reactions. With the inclusion of thermal imaging analysis, this process is supported, and the image from the thermal imager can be objectively analyzed. The method is rapid, non-invasive, and may accompany standard reporting of allergic skin reactions [4].

The thermal imaging image provides information about the size of the inflamed area and its temperature. There are different types of thermal cameras, but FLIR T620 can be used for clinical practice. The analysis is performed using specialized software, as well as comparisons between the reaction to the allergen and reactions to the positive control, negative control, or intact skin area.

After performing the test and standard reading, a thermal camera is taken of the skin areas of interest. The thermal imaging methodology developed by us is different for each of the three skin allergy tests. The image is analyzed by extracting certain (basic) temperature parameters for each zone. Based on them, the additional parameters are calculated, which determine whether the reaction is positive and what is its intensity, which in turn supports the standard reading and minimizes the possibility of misinterpretation of the result.

In this chapter, we will present each of the three allergy tests in turn. Approaches to temperature analysis will be proposed for each test, which will allow quantitative measurement of the calor due to allergic inflammation. The conclusion will summarize the limit values of the indicators and their clinical significance in the reporting of tests.

#### **2. Thermal imaging analysis of allergic skin reactions in prick test**

In the Prick test, two thermal imaging images are taken—one before it is performed and one after it. It is necessary to observe the conditions for thermovisiographic imaging [5]. In standard reading, clinicians touch the skin to sense the

#### *Calorimetry in Allergy Diagnostic DOI: http://dx.doi.org/10.5772/intechopen.102583*

presence and size of the papule. It is necessary to do this after the second scan, so as not to affect the temperature of the examined skin area. Temperature analysis requires measuring the change in each area of the skin. To achieve this, the hand needs to be positioned in the same way in both shots. The stand created by us (**Figure 1**) allows the patient's arm to be placed comfortably, and the curved shape of the two beds for the forearm to stabilize it in a certain position, regardless of its anatomical features. In addition to support elements, the stand includes a plastic template with five holes. The pattern fixation system above the volar surface of the forearm allows the arm to be removed and then repositioned, leaving the pattern position unchanged. The examined five skin areas can be marked with a skin marker after applying the template. The distance between the edges of each hole is 10 mm, enough to prevent the mixing of skin reactions between two adjacent tests (**Figure 2**).

The strongest skin reaction observed after the Prick test is to the positive control. Its size varies between 10 and 30 m**m** [6]. The diameter of the holes in the template is 20 mm, which may not, in some cases, cover the full size of the reaction to a histamine solution. However, this size is large enough for the temperature reactions of the negative control and the tested allergens.

**Figure 1.** *Stand for fixing the patient's forearm during the prick test.*

#### **Figure 2.**

*Placing the patient's hand and outlining three skin areas (depending on the number of tested allergens) in which the prick test will be performed.*

#### **2.1 Analysis of the temperature of the skin areas BEFORE performing a prick test**

Before conducting the test, the places where it will be performed are determined. The initial temperature of the skin areas on which the reactions will be observed should be uniform, without large amplitudes. These are observed in cases of superficially located large blood vessels (v. Cephalica; v. Basilica; v. Mediana anterbrachii). For this reason, it is necessary to initially capture the skin areas and reposition the arm (**Figure 3**). Most studies do not analyze the initial skin temperature, although it is the starting point for the temperature change [6–8]. There were no statistically significant differences in skin temperatures prior to the test when subcutaneous main blood vessels were avoided [9]. If the blood vessel passes through the examined area, it will not allow significant changes in temperature to be reported, regardless of the severity of the allergic inflammation. On the other hand, there is a risk of injury to the blood vessel and compromise of the allergy test [10].

The Prick test is most commonly used to test for sensitization to local anesthetics, foods, and medications. Before the test, two or three anesthetics are selected to be checked for sensitization.

The initial survey gives us information about the following main temperature parameters (**Figure 4**). With the help of specialized software, the skin areas of interest are outlined, and then a table with the temperatures of each of them is displayed.

*X1* – the average temperature of the skin area in which the test allergen will be located before the test.

*Neg1* – the average temperature of the skin area in which the negative control before the test will be located

*Pos1* – the average temperature of the skin area in which the positive control will be located before the test

#### **2.2 Analysis of the temperature of the skin areas AFTER performing a prick test**

After shooting, the test is performed in the standard way [10]. Before proceeding to the standard reading, the hand is carefully placed on the stand and the template is positioned so that the openings coincide with the marked skin areas. The second thermal imaging is done.

**Figure 3.** *Location of subcutaneous main blood vessels.*


#### **Figure 4.**

*First temperature imaging and determination of the temperature in the skin areas where the prick test will be performed.*

*X2* – the average temperature of the skin area where the test allergen will be located after the test.

*Neg2* – the average temperature of the skin area in which the negative control will be located after the test

*Pos2* – the average temperature of the skin area in which the positive control will be located after the test (**Figure 5**).

There are different methods for analyzing the data obtained. Some authors believe that the information obtained from the second image is sufficient to determine whether a reaction is positive or negative [11]. The parameter to be analyzed is only the value of **X2**. The disadvantage of this type of analysis is that they do not include

#### **Figure 5.**

*Second temperature imaging and determination of the temperature in the skin areas in which the prick test was performed.*

the initial skin temperature, which is individual for each patient and may affect the final assessment. The results also show large variations in different patients, so conclusions based on this indicator alone may be wrong.

Another variant found in the literature is to study the change in temperature that has occurred in the skin area of the respective allergen (Eq. (1)) [6, 11, 12].

$$
\Delta X = X\_2 - X\_1 \tag{1}
$$

When performing the test on the patient's back, an increase in the temperature of the positive reactions is reported, which reaches 2.9 degrees in the strongest reactions and is 1 degree in the other positive reactions. In our study on the volar surface of the hand, a slight rise in temperature was observed during the positive control, and in women, in some cases, even a slight cooling of the site was found. The reason for this difference is the place where the test was performed. The forearm during the test is located at a distance from the body and the temperature of her skin drops. The cooling in the negative reactions is significantly greater than the cooling that occurred in the positive control because they do not show inflammation to compensate for it. When the test is performed on the back, where the possibilities of temperature homeostasis of the body do not allow such a strong cooling within 15 minutes, a rise in temperature is observed even in negative controls [11].

The cooling of the negative reactions is more pronounced in women than in men.

While in the field of positive reactions, the differences between the sexes are minimal [13]. The temperature change of the skin in the area of positive control increases in 94% of patients [14]. In such studies, this percentage reached 98% [13].

The temperature rise is significant from 1.5 to 4.00 degrees in some of the studies [6]. While in others the temperature rise in positive reactions is significantly lower: 0.9 � 0.48 degrees [13]. The difference in results is due to the type of allergens used (local anesthetic—Mepivastesin and pollen allergens) and the intensity of the positive reactions.

Allergic inflammation in the negative reactions is absent, so the researchers did not report a change in temperature in them [8, 11, 12].

The change in temperature in most cases is indicative of the presence and strength of a positive reaction to the tested allergen. The analysis should be done according to the patient's gender, that is, variations between men and women are found.

To determine the change in temperature, which is due solely to allergic inflammation, it is necessary to take into account the drop in skin temperature during the test. It varies from patient to patient and depends on individual characteristics, such as subcutaneous tissue, blood supply, and thermal homeostasis. By changing the temperature in the area of the negative control, the cooling of the skin during the test can be monitored Eq. (2):

$$
\Delta \text{Neg} = \text{Neg}\_2 - \text{Neg}\_1 \tag{2}
$$

The difference between the value of *ΔX* and *ΔNeg* is the warming due to allergic inflammation, excluding all other factors influencing the skin temperature. Eq. (3)

$$
\Delta \mathbf{X} \mathbf{a} = \Delta \mathbf{X} - \Delta \mathbf{N} \mathbf{e} \tag{3}
$$

The indicator *ΔX* has values below 0.5C for negative allergic reactions and over 0.5 for positive ones. The stronger the allergic inflammation, the higher its value. When the value is below 0.5 allergic inflammation has no clinical value. It can be considered null and void and explained by the difference in the location of the skin areas in

*Calorimetry in Allergy Diagnostic DOI: http://dx.doi.org/10.5772/intechopen.102583*

which the test was performed. This indicator does not depend on the sex of the patient and allows a comparison of the intensity of allergic reactions between men and women [13].

Of interest are patients with sensitive skin who have dermographism. This is a condition in which the skin reacts by inflammation to nonspecific irritants. Mechanical trauma during the test elicits a response that may be incorrectly reported as allergic [15]. This condition is the reason to include positive and negative controls in the Prick test. In patients with dermographism, measurements of papules and erythema are reported and compared with those of the negative control. **Figure 6** shows three reactions—to anesthetic, to negative control, and to positive control (closest to the patient's tattoo).

All three reactions have papule sizes over 3 mm, which makes it difficult to read the standard. Thermovisiographic analysis also allows to check the temperature changes in the skin areas during the test. Four skin areas are examined (**Figures 7** and **8**):

#### **Figure 6.**

*Reactions after prick test of a patient with dermographism.*


**Figure 7.** *First shot before prick test of a patient with dermographism.*


#### **Figure 8.**

*Second imaging after prick test of a patient with dermographism.*

Ar1 – intact skin area in the area of which there is no legal test.

Ar2 – examination of a local anesthetic (mepivacaine).

Ar3 – negative control.

Ar4 – positive control.

After analyzing the main indicators, the additional ones can be calculated by Eqs. (1)–(3):

$$
\Delta X = X\_2 - X\_1 = 33.7 - 34.5 = -0.8.
$$

$$
\Delta \text{Neg} = \text{Neg}\_2 - \text{Neg}\_1 = 34.2 - 35.1 = -0.9.
$$

$$
\Delta Xa = \Delta X - \Delta \text{Neg} = -0.8 - (-0.9) = 0.1.
$$

The results show that there is no evidence of allergy to the studied local anesthetic. If we make similar calculations for the positive control, we will get a value of *ΔXa* ¼ 1*:*4.

*ΔXa* ¼ (Pos2 – Pos1) - *ΔNeg* = (35.8–35.3) – (�0.9) = 1.4.

The calculations show that the reactions of dermographism are subject to thermal imaging analysis. This is a major advantage over standard reporting in the presence of dermographism. More studies of this type of patient are needed.

The analysis of the results obtained from the thermal imaging provides information about the temperature changes in the skin at the places where the test was performed. By considering various parameters, a better understanding of the inflammation at the sites of allergens and controls is achieved. The calculation of additional parameters based on the results of both controls helps to unify the process and to create limit values that do not depend on the sex of the patient and the individual characteristics of his/her skin.

#### **3. Thermal imaging analysis of skin-allergic reactions in intradermal tests**

The test has many similarities with the Prick test—it is performed on the patient's forearm, areas with superficial blood vessels are avoided. The hand is placed on a

#### *Calorimetry in Allergy Diagnostic DOI: http://dx.doi.org/10.5772/intechopen.102583*

stable base. The allergen is administered intradermally using a syringe and needle. The amount is about 0.02 ml. A papule of about 2–3 mm forms on the skin above the tip of the needle. In addition to the tested allergens, the test is performed with negative and positive control. In this test, it is necessary to avoid areas with superficial main blood vessels. Unlike the Prick test, here the number of allergens is usually significantly higher. Thermal imaging is done after the test. Due to the large number of tests located close to each other, the use of the Prick test stand is not appropriate. On this trichina, fine metal indicators are placed on the hand, which are clearly visible on the thermal image and indicate the place where the test was performed (**Figure 9**).

The thermal imaging image is similar to that of the Prick test, but due to the lack of an armrest with outlined areas, their size and location are determined subjectively depending on the course of the isotherms (**Figure 10**).

*Analysis of the temperature of the skin areas AFTER performing an intradermal test.* The survey provides information on the main temperature parameters:

*Z* – the average temperature of the skin area in which the test allergen is located.

*Neg* – the average temperature of the skin area in which the negative control is located *Pos* – the average temperature of the skin area in which the positive control is located (**Figure 11**)

### As in the Prick test, the analysis here can be based on the absolute temperature value of the reactions [16]. Another approach is to find the difference between the

**Figure 9.** *Intradermal allergy test.*

#### **Figure 10.**

*Thermal imaging image after the intradermal test. Green arrows indicate positive reactions and blue arrows indicate negative reactions.*


**Figure 11.** *Outline the zones of reactions on the thermal image.*

reaction temperature and that of the negative control. The value of allergic inflammation is measured as in the Prick test, but the control area is replaced with the area of negative control (Eq. (4)).

$$
\Delta Z = Z - \text{Neg}\tag{4}
$$

At values of *ΔZ* above 0.6 degrees, the reactions can be considered positive and below 0.6 degrees - negative [13]. A similar comparison can be made with respect to the positive control (Eq. (5))

$$
\Delta Z = Z - \text{Pos} \tag{5}
$$

With the values of *ΔZ* above �1.0 degrees, the reactions can be considered positive and below �1 degree - negative [13].

In intradermal tests, needle pricking results in severe mechanical trauma that causes nonallergic inflammation at the puncture site. For this reason, comparing the changes in temperature in each of the skin areas is not as informative as in the Prick test. Also, the comparison of the temperature of the reactions with those of the negative and positive control gives sufficient information not only about whether the reaction is positive but also what is its intensity. With the help of both controls, the reactivity of the skin can be determined in the absence of allergic inflammation and in case of a strong skin-allergic reaction. The stronger positive reactions show closer temperature values to those of the positive control, while the weaker ones approach more to the negative control.

#### **4. Thermal imaging analysis of skin-allergic reactions after patch test**

In this type of test, the allergens are placed on a hypoallergenic sticker with chambers, which is glued to the patient's back and remains there for 48 hours. The

#### *Calorimetry in Allergy Diagnostic DOI: http://dx.doi.org/10.5772/intechopen.102583*

long period of time presupposes a different approach in the thermal imaging analysis—a photograph is taken of the patient's skin after the patch has been removed and the irritating reaction has passed since its removal. The problem is that the test is done on a large area of skin with underlying muscle groups, tendons, and vertebrae. Like the volar surface of the forearm, the temperature is different in different areas. This makes the use of negative control (empty chamber without hapten in it) unsuitable for temperature analysis, due to the large distance at which it is located relative to allergens. For example, if the empty chamber is located near the spine, the skin temperature in that area will always be lower than the temperature in an area above a muscle, regardless of the presence of an allergic reaction. On the other hand, unlike the Prick test, here the reactions are limited only to the size of the area with which the allergen has been in contact and do not affect **the surrounding skin areas** [17, 18]. Therefore, the correct approach is to compare the temperature of each reaction with the temperature of a nearby skin area that has not been in contact with the allergen.

A template is placed to identify the skin areas that have been in contact with the bitten. With its help, the location of each reaction can be clearly determined, both in standard and thermal imaging (**Figure 12**).

The size of the templates may vary depending on the number of haptens tested (**Figure 13**).

**Figure 12.**

*Thermal imaging of a patient's back after a patch test with a template for thermovisiographic reading. The skin areas of the four allergens and the lateral area used for the control area are marked with arrows.*

**Figure 13.** *Investigated reactions and their respective control areas.*

#### **Figure 14.**

*Different positive reactions (standard and thermal imaging). 1 - slightly (weak) positive; 2 - strongly positive; 3 - extremely positive.*

The standard reading divides the established reactions into several groups—negative, doubtful, weakly positive, strongly positive, and extremely positive (**Figure 14**).

In both the Prick test and the Patch test, different approaches can be used to analyze reactions. Some studies have involved measuring the absolute skin temperature of reactions without comparing control sites. When comparing the average values of the reactions, the negative ones have an average temperature of 34.7°C; weakly positive - 35.1°C, and strongly positive - 35.7°C. At the maximum temperatures, the values are—for the negative reactions - 35.0°С; in the weakly positive - 35.5°С, in the s**trongly positive - 36.0°C** [18]. Despite the seemingly large differences in the values of the different reactions in practice, it is established that the same allergic inflammations can be manifested with different skin temperatures [19]. The main reason for this is the temperature differences on the surface of the skin on the patient's back. Often there are many nonallergic inflammations on the skin, which can be mistaken for positive and even very positive reactions (**Figure 15**).

#### **Figure 15.**

*Thermal imaging of the back without skin inflammations and thermovision imaging of the back with nonallergic skin inflammations on which a patch test was performed.*

**Figure 16.** *Extremely strong positive reaction with vesicles in three of the studied haptens.*

Another approach is to analyze the difference between the skin temperature at the reaction and that of the corresponding control area. To determine the temperature, which is due solely to allergic inflammation *ΔY*, we subtract the temperature of the control area *Ycontrol* from the temperature in the skin area that was in contact with the hapten *Yreaction* (Eq. (6)).

$$
\Delta Y = Y\_{reaction} - Y\_{control} \tag{6}
$$

The indicator *ΔY* with values above 1.6 is considered extremely positive. Values between 0.9 and 1.6 are characteristic of strongly positive reactions. It is difficult to distinguish the weakly positive reactions from the suspicious and negative reactions because in all three *ΔY*, it is below 0.9 degrees. The role of calorimetry in the Patch test is to strongly distinguish between highly positive and extremely positive reactions and to confirm the results of the standard reading.

Of particular interest are the extremely positive reactions in which vesicles form in the center of the skin reaction. They can be easily recognized by the thermal image there is an area with a temperature higher than the surrounding above 1.6 degrees and in its center a few points with a significantly lower temperature corresponding to the vesicles. The bubbles that form are filled with serous fluid, which cools much more easily than the skin and therefore its temperature is much lower (**Figure 16**).

There is no difference in the thermal imaging test in the Patch test for early and late reading. The analysis may include both mean and maximal skin temperature. Of great importance is the choice of control site for each of the reactions. This should be an area close to the reaction area, but not exposed to a hapten. In patients with irritated skin on the back, an area where there is less inflammation should be chosen. This supports both standard and thermal imaging of the results.

#### **5. Conclusion**

The measurement of the temperature of the skin areas in allergic tests gives objective data on the inflammation and its intensity. Each test examines different


#### **Figure 17.**

*Temperature indicators used in the various allergy tests and their limit values.*

indicators, the value of which complements the results of standard reporting and assists clinicians in their practice (**Figure 17**).

In the Prick test, reactions with a value of показа *ΔXa* below 0.5 are negative, while in higher values, they should be considered positive.

In intradermal tests, reactions with показа *ΔZ* (negative control) below 0.6 were negative, while at higher values, they were positive.

In intradermal tests, reactions with показа *ΔZ* (positive control) below �1.0 were negative, while at higher values, they were positive.

In Patch test, reactions with показа *ΔY* below 0.9 are negative or weakly positive; over 0.9 - strongly positive; over 1.6 - extremely positive.

The studies presented in this article have a high clinical value and provide a more complete understanding of the results of allergy tests. The high correlation between the clinical results and the thermal imaging examination (over 95% correlation) are the grounds for applying the method in parallel with the other diagnostic methods in allergology.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Calorimetry in Allergy Diagnostic DOI: http://dx.doi.org/10.5772/intechopen.102583*

#### **Author details**

Evgeni Stanev\* and Maria Dencheva Faculty of Dental Medicine, Sofia, Bulgaria

\*Address all correspondence to: stanev242@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by José Luis Rivera Armenta and Cynthia Graciela Flores Hernández*

Calorimetry is used to measure the transfer and exchange of heat. It is a technique that has applications in different research and industrial sectors. It can be applied in kinetic studies as well as to measure physical changes of first- and second-order transitions such as glass transition, melting, and crystallization. It can also be used to evaluate thermodynamic parameters. This book reports on calorimetry in three sections: "Applications in General", "Calorimetry in Materials", and "Calorimetry in Biotechnology".

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Applications of Calorimetry

Applications of Calorimetry

*Edited by José Luis Rivera Armenta* 

*and Cynthia Graciela Flores Hernández*